Cyclin a sc 596

Protease inhibitor cocktail (P8340), phosphatase inhibitor cocktail 2 (P5726) and 3 (P0044), and dimethyl sulfoxide (D8418) were purchased from Sigma-Aldrich. The primary antibodies used in this study from Santa Cruz biotechnology were cyclin D1 (sc-753); cyclin A (sc-596); cyclin B1 (sc-245). Additionally, we used pHH3ser10 (9701A; Cell signaling), LC-3 (L8918; Sigma), cleaved caspase-3, and PARP (cell signaling) and GAPDH (IMG-5019A-2; IMGENEX). Horseradish peroxidase-conjugated secondary antibodies of goat anti-rabbit (4010-05) and goat anti-mouse (1012-05) were purchased from Southern Biotech.

For Western blot analysis, frozen liver tissue was homogenized, and whole-cell extracts were prepared using NP40 lysis buffer. Total protein lysates (40 µg) were separated by 12.5% SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, and analyzed by immunoblot with the following primary antibodies: Cyclin A (sc596; 1:1000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), PCNA (DLN-06197; 1:1000; Dianova, Hamburg, Germany), NRF2 (16396-1-AP; 1:1000; Proteintech, Rosemont, IL, USA) and GAPDH (MCA4739; 1:5000; Bio-Rad Laboratories). Secondary antibodies were HRP-linked anti-rabbit IgG (7074; 1:5000; Cell Signaling Technology) and HRP-linked anti-mouse IgG (sc-516102; 1:5000; Santa Cruz Biotechnology). The labeled proteins were visualized using enhanced chemiluminescence (RPN2232; Merck Millipore) and the resulting light emission was detected by a luminescent image analyzer ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA).